The use of antibodies to block the activity of foreign and/or endogenous polypeptides provides an effective and selective strategy for treating the underlying cause of disease. In particular is the use of monoclonal antibodies (MAb) as effective therapeutics such as the FDA approved ReoPro (Glaser, V. (1996) Can ReoPro repolish tarnished monoclonal therapeutics? Nat. Biotechnol. 14:1216-1217), an anti-platelet MAb from Centocor; Herceptin (Weiner, L. M. (1999) Monoclonal antibody therapy of cancer. Semin. Oncol. 26:43-51), an anti-Her2/neu MAb from Genentech; and Synagis (Saez-Llorens, X. E., et al. (1998) Safety and pharmacokinetics of an intramuscular humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. Pediat. Infect. Dis. J. 17:787-791), an anti-respiratory syncytial virus MAb produced by Medimmune.
Standard methods for generating MAbs against candidate protein targets are known by those skilled in the art. Briefly, rodents such as mice or rats are injected with a purified antigen in the presence of adjuvant to generate an immune response (Shield, C. F., et al. (1996) A cost-effective analysis of OKT3 induction therapy in cadaveric kidney transplantation. Am. J. Kidney Dis. 27:855-864). Rodents with positive immune sera are sacrificed and splenocytes are isolated. Isolated splenocytes are fused to melanomas to produce immortalized cell lines that are then screened for antibody production. Positive lines are isolated and characterized for antibody production. The direct use of rodent MAbs as human therapeutic agents were confounded by the fact that human anti-rodent antibody (HARA) responses occurred in a significant number of patients treated with the rodent-derived antibody (Khazaeli, M. B., et al., (1994) Human immune response to monoclonal antibodies. J. Immunother. 15:42-52). In order to circumvent the problem of HARA, the grafting of the complementarity determining regions (CDRs), which are the critical motifs found within the heavy and light chain variable regions of the immunoglobulin (Ig) subunits making up the antigen-binding domain, onto a human antibody backbone found these chimeric molecules are able to retain their binding activity to antigen while lacking the HARA response (Emery, S.C., and Harris, W. J. “Strategies for humanizing antibodies” In: ANTIBODY ENGINEERING C. A. K. Borrebaeck (Ed.) Oxford University Press, N.Y. 1995. pp. 159-183. A common problem that exists during the “humanization” of rodent-derived MAbs (referred to hereon as HAb) is the loss of binding affinity due to conformational changes in the three-dimensional structure of the CDR domain upon grafting onto the human Ig backbone (U.S. Pat. No. 5,530,101 to Queen et al.). To overcome this problem, additional HAb vectors are usually needed to be engineered by inserting or deleting additional amino acid residues within the framework region and/or within the CDR coding region itself in order to recreate high affinity HAbs (U.S. Pat. No. 5,530,101 to Queen et al.). This process is a very time-consuming procedure that involves the use of expensive computer modeling programs to predict changes that may lead to a high affinity HAb. In some instances the affinity of the HAb is never restored to that of the MAb, rendering them of little therapeutic use.
Another problem that exists in antibody engineering is the generation of stable, high-yielding producer cell lines that is required for manufacturing of the molecule for clinical materials. Several strategies have been adopted in standard practice by those skilled in the art to circumvent this problem. One method is the use of Chinese Hamster Ovary (CHO) cells transfected with exogenous Ig fusion genes containing the grafted human light and heavy chains to produce whole antibodies or single chain antibodies, which are a chimeric molecule containing both light and heavy chains that form an antigen-binding polypeptide (Reff, M. E. (1993) High-level production of recombinant immunoglobulins in mammalian cells. Curr. Opin. Biotechnol. 4:573-576). Another method employs the use of human lymphocytes derived from transgenic mice containing a human grafted immune system or transgenic mice containing a human Ig gene repertoire. Yet another method employs the use of monkeys to produce primate MAbs, which have been reported to lack a human anti-monkey response (Neuberger, M., and Gruggermann, M. (1997) Monoclonal antibodies. Mice perform a human repertoire. Nature 386:25-26). In all cases, the generation of a cell line that is capable of generating sufficient amounts of high affinity antibody poses a major limitation for producing sufficient materials for clinical studies. Because of these limitations, the utility of other recombinant systems such as plants are currently being explored as systems that will lead to the stable, high-level production of humanized antibodies (Fiedler, U., and Conrad, U. (1995) High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Bio/Technology 13:1090-1093).
Other factors that naturally contribute to antibody diversity are the phenomena of class switch recombination (CSR) and somatic hypermutation. Class switch recombination is a region-specific recombination at the DNA level that results in the substitution of one immunoglobulin heavy chain region for another. Somatic hypermutation is the name of the phenomenon in which fully assembled immunoglobulin genes nevertheless undergo mutation in the variable regions only. Somatic hypermutation is thought to promote affinity maturation in antibodies.
An enzyme that has been found to play a critical role in both CSR and somatic hypermutation is activation-induced cytodine deaminase (“AID” or “AICDA”). Muramatsu et al. cloned the murine AID (SEQ ID NOs:43 and 44) (Muramatsu et al. (1999) J. Biol. Chem. 274(26):18470-18476), while the human AID (SEQ ID NOs:39 and 40) was cloned by Muto et al. (2000) Genomics 68:85-88). The mouse and human AID share 92% identity at the amino acid level, both containing 198 amino acids with a conserved cytodine deaminase motif. It is believed that AID acts to induce lesions in the DNA (i.e., deamination of deoxycytidines leading to dU/dG pairs) (Petersen-Mahrt et al. (2002) Nature 418:99-104). AID appears to be expressed only in stimulated B cells in germinal centers (Okazaki et al. (2002) Nature 416:340-345), and appears to be responsible for both CSR (Petersen et al. (2001) Nature 414:660-665) and somatic hypermutation (Yoshikawa et al. (2002) Science 296:2033-2036).
Revy et al. showed that human patients with a defect in the AID gene (hyper IgM syndrome, or HIGM2) lacked both CSR and somatic hypermutation activity (Revy et al. (2000) Cell 102:565-575). Similarly, spleen cells from AID−/− mice failed to undergo somatic hypermutation or CSR when stimulated in vitro (Muramatsu et al. (2000) Cell 102:553-563).
A method for generating diverse antibody sequences within the variable domain that results in HAbs and MAbs with high binding affinities to antigens would be useful for the creation of more potent therapeutic and diagnostic reagents respectively. Moreover, the generation of randomly altered nucleotide and polypeptide residues throughout an entire antibody molecule will result in new reagents that are less antigenic and/or have beneficial pharmacokinetic properties.